Biosynthesis of Panaxynol and Panaxydol in Panax Ginseng
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Molecules 2013, 18, 7686-7698; doi:10.3390/molecules18077686 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Biosynthesis of Panaxynol and Panaxydol in Panax ginseng Nihat Knispel 1,†, Elena Ostrozhenkova 1,†, Nicholas Schramek 1, Claudia Huber 1, Luis M. Peña-Rodríguez 1,‡, Mercedes Bonfill 2, Javier Palazón 2, Gesine Wischmann 3, Rosa M. Cusidó 2,* and Wolfgang Eisenreich 1,* 1 Lehrstuhl für Biochemie, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany 2 Laboratorio de Fisiología Vegetal, Facultad de Farmacia, Universidad de Barcelona, 08028 Barcelona, Spain 3 FloraFarm, Bockhorn, 29664 Walsrode-Bockhorn, Germany † These authors contributed equally to this work. ‡ On sabbatical leave from Centro de Investigación Científica de Yucatán, Mérida, México * Authors to whom correspondence should be addressed; E-Mails: [email protected] (W.E.); [email protected] (R.M.C.); Tel.: +49-89-289-13336 (W.E.); +34-93-402-0267 (R.M.C.) Received: 6 May 2013; in revised form: 13 June 2013 / Accepted: 28 June 2013 / Published: 2 July 2013 Abstract: The natural formation of the bioactive C17-polyacetylenes (−)-(R)-panaxynol and panaxydol was analyzed by 13C-labeling experiments. For this purpose, plants of 13 Panax ginseng were supplied with CO2 under field conditions or, alternatively, sterile 13 root cultures of P. ginseng were supplemented with [U- C6]glucose. The polyynes were isolated from the labeled roots or hairy root cultures, respectively, and analyzed by quantitative NMR spectroscopy. The same mixtures of eight doubly 13C-labeled isotopologues and one single labeled isotopologue were observed in the C17-polyacetylenes obtained from the two experiments. The polyketide-type labeling pattern is in line with the biosynthetic origin of the compounds via decarboxylation of fatty acids, probably of crepenynic acid. The 13C-study now provides experimental evidence for the biosynthesis of panaxynol and related polyacetylenes in P. ginseng under in planta conditions as well as in 13 root cultures. The data also show that CO2 experiments under field conditions are useful to elucidate the biosynthetic pathways of metabolites, including those from roots. Molecules 2013, 18 7687 Keywords: Panax ginseng; Araliaceae; panaxynol; panaxydol; falcarinol; polyyne; 13 crepenynic acid; isotopologue profiling; CO2 1. Introduction Extracts of ginseng (Panax ginseng C.A. Meyer) roots are used as health promoting drugs in traditional Oriental medicine. In recent times, however, ginseng has also gained importance in Western medicine as an anti-aging drug with an increasing market value [1]. Although the mechanisms of action of ginseng on human metabolism and health are not well understood, bioactivity is mainly assigned to the presence of ginsenosides, a group of secondary metabolites belonging to the triterpene saponins class [2–4]. However, additional bioactive natural products are present in the extracts of P. ginseng that contribute to the overall effect of ginseng. Among these bioactive metabolites, the C17-polyacetylenes, which include panaxynol (1, Figure 1) and its related epoxide panaxydol (2), have attracted remarkable interest mainly due to their biological activities [5]. Panaxynol was first isolated from roots of P. ginseng C.A. Meyer and described in 1964 [6]. To date, more than 16 polyacetylenes have been reported from P. ginseng [7] and other plants, mainly from the Araliaceae and Apiaceae families, including carrots, parsnip, parsley, fennel and celery [8,9]. Figure 1. Structures of (−)-(R)-panaxynol (1) and panaxydol (2). Panaxynol and related polyynes have shown cytotoxic activity against several human tumour cell lines in vitro [10–15]. In vivo studies have confirmed the high potential of these metabolites for antitumour treatment [13]. Panaxynol-type polyacetylenes also exhibit significant antimicrobial (e.g., antimycobacterial) [16], antifungal [17–19], antiplatelet and anti-inflammatory [20–23], neuroprotective [24,25], antimutagenic [26–28], antiproliferative [12,21,29,30], antitrypanosomal [4], allergenic and skin-irritating activities [31–34]. The broad bioactivity of these metabolites, in combination with their high potential to benefit human health, reflects the importance of these polyacetylenes and the need for more detailed studies of their biosynthetic route, as a prerequisite to perhaps produce them by biotechnological means (e.g., using modified plants or recombinant microbial cultures). On the basis of their structural similarity to fatty acids and of early experiments with radiolabeled fatty acids, it is widely accepted that the linear C17 polyacetylenes are derived from C18 unsaturated fatty acids [35–37] (reviewed in [9]). It has also been proposed, without experimental validation, that Molecules 2013, 18 7688 3-hydroxyoleic acid could serve as an intermediate in panaxynol biosynthesis [36] and that aryl polyacetylenes are derived from the shikimate pathway [9]. However, the experimental evidence for the fatty acid route leading to C17 polyacetylenes is rather weak due to low incorporation rates of the radiolabeled precursors into the final products and the question remains open whether the fatty acid route is the main and only biosynthetic pathway leading to these secondary metabolites. In this study, 13 13 we have used CO2 and C-labeled glucose as tracers for in vivo isotope labeling of P. ginseng plants and root cultures, respectively, to elucidate the biosynthetic pathway of C17 polyacetylenes. 2. Results and Discussion 2.1. Isolation and Identification of Panaxynol (1) and Panaxydol (2) 13 13 Lyophilized roots from plants treated with CO2 or root cultures enriched with [U- C6]glucose were extracted with hexane. Purification of the corresponding extracts using column chromatography yielded the less polar panaxynol (1) and more polar panaxydol (2) in pure form; both metabolites were identified by comparing their spectroscopic data (1H and 13C-NMR) to those reported in the literature [38–41]. However, in view of the conflicting reports on the structures of this type of polyacetylenes (e.g., their stereoconfigurations), it is important to emphasize the correct identification of the isolated panaxynol; this metabolite was originally reported by Takahashi from P. ginseng [6] and later reported with the names falcarinol from Falcaria vulgaris [42] and carotatoxin from Daucus carota [43]. The first attempt to establish the absolute stereochemistry at C-3 of the compound was carried out by Larsen et al. [44], who described falcarinol from Seseli gummiferum as having a 3-(R) chirality on the basis of chemical correlation studies. The second attempt was carried out by Shim et al. [45,46] who described panaxynol as having a 3-(S) chirality on the basis of CD measurements. More recently, modified Mosher’s methods have described falcarinol from Dendropanax arboreus as being dextrorotatory and having the 3-(S) chirality [47], whereas panaxynol from P. ginseng was reported as being levorotatory and having the 3-(R) chirality [40]. These later reports were confirmed by Zheng et al. [48] who carried out the enantiospecific synthesis of the two isomers of falcarinol/panaxynol and demonstrated that the 3-(R) and 3-(S) chiralities correspond to the levorotatory and dextrorotatory enantiomers, respectively. The negative value of the optical activity of panaxynol obtained in this study also indicated its 3-(R) chirality. 2.2. Biosynthesis of Panaxynol and Panaxydol in P. ginseng 13 2.2.1. In planta Experiments with CO2 13 Experiments with CO2 best resemble the physiological conditions for plants and the labeling profiles in the biosynthetic products represent quasi undisturbed in planta conditions. More specifically, the results obtained from these experiments are free from artifacts due to metabolic stress reactions (e.g., triggered by wounding in labeling experiments with cut plant organs) or due to the usage of non-physiological substrates in experiments with cell cultures. The strategic idea behind 13 13 isotopologue profiling using CO2 is the photosynthetic generation of completely C-labeled metabolic intermediates (e.g., triose and pentose phosphates and products thereof) during an incubation Molecules 2013, 18 7689 13 period with CO2 (pulse period). During a subsequent chase period, the plants are allowed to grow 12 under standard conditions (i.e., in a natural atmosphere with CO2) for several days in which unlabeled photosynthetic intermediates are generated (i.e., with 12C). These 13C- and 12C-intermediates from the pulse and the chase periods, respectively, are then taken by the plant as precursors for downstream biosynthetic processes. Consequently, the combination of these precursor units results in specific mixtures of 13C-isotopologues in the product. In other words, mixtures of unlabeled and multiple 13C-labeled isotopologues are generated as a consequence of the biosynthetic history of the metabolites under study. Using quantitative NMR spectroscopy, these isotopologue profiles can be assigned and attributed to biosynthetic pathways. Several recent examples have demonstrated the power of this experimental approach [49–51]. 13 13 CO2-labeling experiments of P. ginseng (Figure 2) were carried out using a portable CO2 unit [52]. 13 To this aim, six-year-old plants of P. ginseng growing under field conditions were exposed to a CO2 atmosphere for 9.5 h and then allowed to grow for 19 days under natural conditions. Extraction of the roots yielded a mixture which,